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J. Comp. Path. 2001, Vol.

124, 7078
doi:10.1053/jcpa.2000.0430, available online at on
Expression of Cytokeratins and Vimentin in
Normal and Neoplastic Tissue from the Bovine
Female Reproductive Tract
C. Pe rez-Mart nez, R. A. Garc a-Ferna ndez, A. Escudero,
M. C. Ferreras and M. J. Garc a-Iglesias
Histology and Pathological Anatomy Section, Department of Animal Pathology: Animal Medicine,
Faculty of Veterinary Science, University of Leon, 24071 Leon, Spain
The distribution of cytokeratins (CKs) and vimentin in the normal genital tract of calves and cows at dierent
stages of the oestrous cycle and in epithelial tumours of the tract was studied immunohistochemically. Few
dierences in CK and vimentin immunolabelling were detected in relation to age or stage of the oestrous
cycle. Coexpression of CKs in simple epithelia and in basal cells of stratied epithelia was detected in the
oviduct and endocervix; this coexpression was dierent from that previously described in women. The
demonstration of CKs but not vimentin in the neoplastic cells of a serous supercial ovarian papilloma
suggested an origin from the ovarian surface epithelium, while the coexpression of CKs and vimentin in
serous papillary and mucinous cystadenomas pointed to a possible origin from the rete ovarii. Studies on
three uterine adenocarcinomas and the ovarian metastases from two of these showed an endometrial-CK
phenotype. The intermediate lament prole of normal endometrium, conserved in uterine adeno-
carcinomas and their ovarian metastases, may be useful in discriminating between ovarian metastases
from endometrial carcinomas and those originating from primary carcinomas in other organs.
2001 Harcourt Publishers Ltd
Introduction tract have received considerable study (Uhrin,
1983a, b; Blazquez et al., 1987), but little is known
Intermediate laments (IFs) represent a large and
about the expression of IFs in the normal genital
diverse family of cytoplasmic proteins (Steinert et
tract, other than the results of a few studies on the
al., 1984) which can be used to dene dierent cell
ovary (Ricken et al., 1995; Van den Hurk et al.,
phenotypes (Franke et al., 1982). Cell typing with
1995). No studies of IF expression in bovine genital
IF antibodies is useful in tumour diagnosis because
tumours have been recorded.
the expression of certain IF proteins is conserved
The aims of the present study in cattle were to
after transformation of cells and can be used to
identify the CK and vimentin immunolabelling
dene the state of dierentation of normal and
patterns in normal female genital organs at dierent
neoplastic cells (Osborn and Weber, 1983). Cyto-
stages of the oestrous cycle, and in genital epithelial
keratin (CK) and vimentin proteins in the genital
tumours, in an attempt to clarify the histogenesis
tract have been studied immunohistochemically in
of such neoplasms and to distinguish ovarian car-
human beings but in only a few mammalian species,
cinomas that have arisen in the genital tract from mainly laboratory animals (Kress et al., 1995; Chat-
those due to metastasis from primary carcinomas eau and Boehm, 1996; Pan and Auersperg, 1998).
The structural features of the bovine female genital in other sites.
00219975/01/010070+09 $3500 2001 Harcourt Publishers Ltd
CKs and Vimentin in the Bovine Genital Tract 71
Materials and Methods with haematoxylin. The immunoperoxidase re-
action was scored (Van Niekerk et al., 1993) as
Normal Genital Tract Study
follows: +, strongly positive epithelial cells 10
100%; +/, strongly positive epithelial cells Samples of ovary, oviduct (ampulla) and uterus
(horn and endocervix) from normal slaughtered <10%; , > 10% of epithelial cells positive, but
with weak immunoreactivity. Swiss Brown cows aged 914 years (n=3 in met-
oestrus; n=3 in dioestrus, n=3 in prooestrus), and
three calves (pre-pubic) aged 45 months were
xed in 10% neutral buered formalin, dehydrated,
and embedded in paran wax. Sections (3 m)
Immunohistochemical Features of Normal Bovine Female
were stained with haematoxylin and eosin (HE)
Genital Tract
and by the Grocott-Gomori methenamine silver
The IF results in normal ovarian cells from the
method (Cook, 1994) to establish the stage of the
surface epithelium and rete ovarii (elements from
oestrous cycle, taking into account particularly the
which epithelial ovarian tumours might arise) are
histological features of the corpora lutea (Mart nez-
recorded in Table 2. The main ndings were (a)
Rodr guez et al., 1987).
the coexpression of CKs (Fig. 1) and vimentin (Fig.
2) in rete ovarii, and (b) the positive reaction for
Tumour Study
CKs (Fig. 3) but negative reaction for vimentin
(Fig. 4) in surface epithelium.
The reproductive tracts of 1489 female cattle
In the oviductal and endocervical epithelia, no
slaughtered in dierent abattoirs in Leo n (Spain)
qualitative cyclical changes in the expression of
were examined over a 4-year period for the pres-
the IFs examined were found in cows. However,
ence of neoplastic lesions. The animals, which had
quantitative changes were observed in the ex-
unknown breeding histories, were either Friesians,
pression of vimentin in these epithelia, fewer posi-
Swiss Browns, Alistano-Sanabresas (an autoch-
tive cells being found in the luteal phase (Tables 3
thonous breed of the region) or cross-bred. Their
and 4). In all animals examined, regardless of
ages ranged from 3 months to 20 years. The histo-
age, reserve cells on the basal membrane of the
pathological diagnoses of the tumours found were
endocervical simple epithelium were identied by
reported previously (Garc a-Iglesias et al., 1991,
immunolabelling for CKs (Fig. 5) and vimentin
1995) as primary uterine adenocarcinomas (n=
(Table 4). Their number varied depending on the
3) and their ovarian metastases (n=2), supercial
stage of the oestrous cycle, with a clear increase in
serous papilloma (n=1), serous papillary cyst-
metoestrus. In all animals, these reserve cells and
adenomas (n=2) and mucinous cystadenoma (n=
the oviductal epithelium showed coexpression of
CKs typical of the basal cells of stratied epithelia
and CKs of simple epithelia.
In the endometrium, the main dierences found
between cows and calves were (1) the presence of
Representative formalin-xed tissue sections (3 m)
CK14 labelling in endometrial epithelium of calves
from the normal tissue and tumour samples were
but not in adult animals, and (2) a lower number
subjected to immunohistochemical labelling with
of CK8,18-positive cells in cows than in calves
an avidinbiotinperoxidase complex (ABC)
(Table 5).
method to examine the changes in CK and vi-
mentin expression patterns. All primary antibodies
were labelled with the Peroxidase Standard Vecta-
Immunohistochemical Features of Epithelial Ovarian and
stain ABC Kit (Vector Laboratories, Burlingame,
Uterine Tumours
CA, USA) except CAM 52 and AF 129, which
were labelled with the Elite Vectastain ABC Kit Table 6 presents the results of the distribution of the
IF proteins in primary epithelial ovarian and uterine (Vector Laboratories). Table 1 shows the working
dilutions and the specicities of the primary anti- tumours, together with ovarian metastases. When
the CK and vimentin expression of epithelial bodies used. Diaminobenzidine tetrahydrochloride
(DAB Substrate Kit; Vector Laboratories) was used ovarian tumours was compared with that in normal
ovarian tissues, the serous supercial papilloma ex- as the substrate-chromogen solution. Negative and
positive controls for the primary and secondary pressed a positive CK phenotype (Fig. 6) and neg-
ative vimentin phenotype (Fig. 7); this suggested that antibodies were included. After development of the
immunoreaction, the slides were counterstained it arose from ovarian surface epithelium, which
C. Pe rez-Mart nez et al. 72
Table 1
Primary antibodies used for immunohistochemical labelling
Clone Character/ Specicity Antigen Dilution Source
species unmasking
AE1/AE3 mo/M CK 18,10,1416,19 T 1 in 200 Zymed Lab. Inc., San Francisco, USA
AF 109 po/Rb CK 1 H 1 in 500 Berkeley Antibodies Co, California, USA
AF 138 po/Rb CK 5 H 1 in 10 000 Berkeley Antibodies Co, California, USA
MK6 po/Rb CK 6 H 1 in 1000 Berkeley Antibodies Co, California, USA
LDS-68 mo/M CK7 H 1 in 75 Sigma Chemical Co., St. Louis, USA
CAM 52 mo/M CK8,18 H 1 in 10 Becton Dickinson, San Jose, USA
MK10 po/Rb CK 10 T 1 in 300 Berkeley Antibodies Co, California, USA
AF 129 po/Rb CK 13 T 1 in 500 D. R. Roop
AF 64 po/Rb CK 14 H 1 in 100 Berkeley Antibodies Co, California, USA
VIM 132 mo/M Vimentin H 1 in 1000 Sigma Chemical Co., St. Louis, USA
mo, Monoclonal; po, polyclonal; CK, cytokeratin; M, mouse; Rb, rabbit.
D. R. Roop, Baylor College of Medicine, Houston, Texas, USA.
T, Trypsin digestion; H, heat pre-treatment (according to Auld, 1994).
Table 2
Immunohistochemical detection of CKs and vimentin in normal bovine ovary
Ovarian Antigen
PanCK CK1 CK5 CK6 CK7 CK8,18 CK10 CK13 CK14 Vimentin
Surface + + + + +
Rete ovarii + + + + +
+, Strongly positive cells 10100%; , weakly positive cells 10100%.
PanCK, clone AE1/AE3.
Fig. 1. Immunolabelling with anti-PanCK antibody in normal
Fig. 2. Immunolabelling with anti-vimentin antibody in nor-
rete ovarii of a cow. ABC method. 250.
mal rete ovarii of a cow. ABC method. 250.
showed the same pattern of IF expression (Figs 3 and
4). In addition, scattered neoplastic epithelial cells serous papillary (Figs 8 and 9) and mucinous
cyst-adenomas resembled that of the rete ovarii (Figs reacted positively for CK13 (Table 6). On the other
hand, the coexpression of CKs and vimentin in the 1 and 2), supporting an origin from this ovarian
CKs and Vimentin in the Bovine Genital Tract 73
structure. Furthermore, when the patterns of CK
and vimentin expression in the serous papillary cyst-
adenomas were compared with those of the other
normal genital organs examined, this type of tumour
showed a phenotype essentially identical with that
of the oviductal epithelium, although the tumour
cells showed negative immunoreactivity for CK14
and positive immunolabelling for CK13 (Table 6).
With regard to the ovarian mucinous cystadenoma,
the tumour basal cells (Fig. 10) showed an IF protein
expression pattern which resembled that of the en-
docervical reserve cells (Fig. 5). The only dierences
between these tumour basal cells and the reserve
cells were the positive immunoreactivity for CK13
and the negative immunolabelling for CK14 in the
tumour cells. An additional histological nding was
the presence of metaplastic foci in the epithelium
Fig. 3. Normal ovarian surface epithelium of a cow showing
that covered the cysts of the mucinous cystadenoma. PanCK-positive cells. ABC method. 250.
These foci showed coexpression of CKs and vi-
mentin (Table 6).
The prole of CKs and vimentin in the uterine
adenocarcinomas was essentially identical with that
of the endometrial epithelium of calves, except
for CK7 and CK13. However, the tumour cells
displayed enhanced expression of CK6 by com-
parison with the normal endometrium of calves;
this resembled the CK6 expression of normal cows.
In addition, less than 10% of tumour cells expressed
CK13 (Table 6). In general, the ovarian metastases
conserved the expression pattern of CKs detected
in their primary uterine adenocarcinomas; there
was, however, greater CK7 expression (Table 6) in
ovarian metastases (Fig. 11) than in primary uterine
adenocarcinoma (Fig. 12).
This study describes the prole of CKs and vi-
Fig. 4. Negative vimentin immunoreactivity in ovarian surface
mentin in normal epithelia of the genital tract from epithelium of a cow. ABC method. 250.
calves and cows and in epithelial tumours of the
Table 3
Immunohistochemical detection of CKs and vimentin in normal bovine oviductal epithelium
Number of cattle (in groups of 3) showing the stated antigen
Groups of
cattle PanCK CK1 CK5 CK6 CK7 CK8,18 CK10 CK13 CK14 Vimentin
Pre-pubic +3 0 +3 +3 +3 +2 0 0 +3 +3
Metoestrus +3 0 +3 +3 +3 +3 0 0 +2 +/2
Dioestrus +3 0 +3 +3 +3 +2 0 0 +3 +/3
Prooestrus +3 0 +2 +3 +3 +3 0 0 +3 +3
+, Strongly positive epithelial cells 10100%; +/, strongly positive epithelial cells <10%.
PanCK, clone AE1/AE3.
C. Pe rez-Mart nez et al. 74
Table 4
Immunohistochemical detection of CKs and vimentin in normal bovine endocervical epithelium
Number of cattle (in groups of 3) showing the stated antigen
Groups of
cattle PanCK CK1 CK5 CK6 CK7 CK8,18 CK10 CK13 CK14 Vimentin
Pre-pubic +3 0 +3 +3 +3 +2 0 0 +3 +/3
Metoestrus +3 0 +3 +2 +2 +3 0 0 +3 +/2
Dioestrus +3 0 +3 2 +2 +3 0 0 +2 +/3
Prooestrus +3 0 +3 +2 +2 +3 0 0 +2 +2
+, Strongly positive epithelial cells 10100%; +/, strongly positive epithelial cells <10%; , weakly positive epithelial cells 10100%.
PanCK, clone AE1/AE3.
Reserve cells and other epithelial cells.
Reserve cells only.
Table 5
Immunohistochemical detection of CKs and vimentin in normal bovine endometrial epithelium
Number of cattle (in groups of 3) showing the stated antigen
Groups of
cattle PanCK CK1 CK5 CK6 CK7 CK8,18 CK10 CK13 CK14 Vimentin
Pre-pubic +3 0 0 3 +/3 +2 0 0 +3 0
Metoestrus +3 0 0 +3 +/2 +/3 0 0 0 0
Dioestrus +3 0 0 +3 +/3 +/2 0 0 0 0
Prooestrus +3 0 0 +3 +/1 +/2 0 0 0 0
+, Strongly positive epithelial cells 10100%; +/, strongly positive epithelial cells <10%; , weakly positive epithelial cells 10100%.
PanCK, clone AE1/AE3.
Table 6
Immunohistochemical detection of CKs and vimentin in bovine epithelial genital tumours
Tumours in
ovary uterus
Antigen Serous Mucinous Serous Ovarian Adenocarcinoma
supercial cystadenoma papillary metastasis
papilloma cystadenoma of uterine carcinomas
PanCK +1/1 +1/1 +2/2 +2/2 +3/3
CK1 0/1 0/1 0/2 0/2 0/3
CK5 0/1 +1/1 +2/2 0/2 0/3
CK6 +1/1 +1/1 +2/2 +1/2; +/1/2 +3/3
CK7 +1/1 +1/1 +2/2 +2/2 +/1/3
CK8,18 +1/1 +/1/1 +2/2 +2/2 +2/3
CK10 0/1 +/1/1 0/2 0/2 0/3
CK13 +/1/1 +1/1 +2/2 +/1/2 +/2/3
CK14 +1/1 +1/1 0/2 +1/2 +3/3
Vimentin 0/1 +1/1 +2/2 0/2 0/3
Values are the number of positive cases over the total number of each type of tumour studied.
+, Strongly positive tumour cells 10100%; +/, strongly positive tumour cells <10%.
PanCK, clone AE1/AE3.
Basal tumour cells.
Other tumour epithelial cells.
Metaplastic foci.
CKs and Vimentin in the Bovine Genital Tract 75
Fig. 5. Positive CK5 immunoreactivity in normal endocervical
reserve cells of a cow. ABC method. 500.
Fig. 7. Negative vimentin immunoreactivity in serous super-
cial papilloma of the bovine ovary. ABC method.
Fig. 6. Serous supercial papilloma of the bovine ovary show-
ing PanCK-positive cells. ABC method. 250.
tract. No relationship was observed between the
Fig. 8. Serous papillary cystadenoma of the bovine ovary
IF expression patterns and the stages of the oestrous
showing PanCK-positive cells. ABC method. 250.
cycle. This accords with the observation that cyclic
morphological changes in cows are slight as com-
pared with those in primates (Salisbury et al., 1982).
The coexpression of simple epithelial markers not been previously identied in cattle. The con-
stant moderate increase in their number in met- (CK7, CK8, CK18) together with markers of basal
cells (CK5, CK14), reported here in bovine ovi- oestrus, and the lack of evident metaplastic
morphological changes, may indicate that these ductal and endocervical epithelia, has been ob-
served in the human male genital tract but not in cells are undergoing cyclical rather than meta-
plastic changes. the human oviduct or endocervix, in which only
simple epithelial CKs are expressed (Quinlan et al., Contrary to the ndings in the human ovary
(Van Niekerk et al., 1993, 1997), but in accordance 1985). On the other hand, reserve cells of the
endocervix have been described in women, and an with previous ndings in cows (Van den Hurk et
al., 1995) and dogs (Vos et al., 1989), the bovine increase in number has been related to a meta-
plastic process (Burghardt, 1973). However, to the ovarian surface epithelium did not show a positive
vimentin reaction. In this respect, the prole of authors knowledge, endocervical reserve cells have
C. Pe rez-Mart nez et al. 76
Fig. 11. Ovarian metastasis from primary uterine adeno-
Fig. 9. Immunolabelling with anti-vimentin antibody of a
carcinoma. Most tumour cells show CK7 im-
serous papillary cystadenoma of the bovine ovary.
munolabelling. ABC method. 250.
ABC method. 250.
Fig. 10. Positive CK5 immunoreactivity in tumour basal cells
of the ovarian mucinous cystadenoma. Note the ab-
sence of a positive immunoreaction in upper layers.
ABC method. 500.
Fig. 12. The CK7-positive tumour cells in this primary uterine
adenocarcinoma are fewer than in Fig. 11. ABC
method. 250.
CKs and vimentin in a serous supercial papilloma
of the ovary, a tumour reported in women (Serov
et al., 1973) and described for the rst time in the originate from the modied coelomic mesothelial
cells covering the ovary. Some authors (Van Nie- cow by Garc a-Iglesias et al. (1991), pointed to
origin from the ovarian surface epithelium. In kerk et al., 1993; Crum, 1994) have proposed that
the rst stage of development of such tumours is the this tumour, additional expression of CK13 (non-
keratinizing squamous epithelial marker) was dem- entrapment of surface epithelium within ovarian
stroma. Based on the occurrence of both CKs and onstrated in only <10% of positive tumour cells, an
immunolabelling pattern related to the progression vimentin in these epithelial tumours and in the rete
ovarii but not in ovarian surface epithelium, we from normal to malignant cells in other tumours
(Xu et al., 1995; Klein-Szanto, 1997). propose a second possible origin from the rete ovarii
for serous papillary and mucinous cystadenomas in It is assumed that, in human beings (Berek, 1989)
and other animal species (Nielsen and Kennedy, female cattle. Regardless of the origin of these
ovarian epithelial tumours, our data conrm that 1990), ovarian serous and mucinous cystadenomas
CKs and Vimentin in the Bovine Genital Tract 77
bovine reproductive tract caudal to the cervix. I. The
in adult animals the ovarian structures derived
vestibule and associated glands. II. The vagina and
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(Crum, 1994). In this respect, the prole of IFs of
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Received, July 30th, 1999

Accepted, July 5th, 2000

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